How cells determine their size is unknown. A recently described adder model for cell size homeostasis transforms prior textbook models for cell size control (i.e. the timer and sizer models, which assumes cells actively monitor time between cell cycles or measure the absolute size of the cell, respectively). The adder model quantitatively spans over 1 billion years of evolutionary divergence to include both Gram?negative Escherichia coli and Gram?positive Bacillus subtilis. Therefore, any mechanism driving the quantity of mass added between cell divisions must be robust to the genetic, molecular, and physiologic difference between these species. The two candidates underlying the mechanism are the chromosome and the proteome (the set of all proteins in the cell), and it is important to understand how the two are coupled during growth. The genome is unique among cellular materials in that a single copy, essentially a single molecule, must be endowed to each descendent cell at division. The proteome must be composed so that the growth of the cell is optimized for a given growth condition and control of the cell cycle. The project employs multi?disciplinary approach ranging from modern microbiological genetic techniques for genome engineering to high?throughput measurements of the composition of the proteome, and connect them via quantitative modeling. More specifically, modern genetic recombineering techniques will be used to progressively increase the size of the E. coli chromosome with foreign, non?coding DNA to double the native size of the chromosome. Using microfluidic and imaging techniques previously used to discover the adder principle, the mass added between divisions will be measured at the single-cell level to establish a direct correlative and causative lik between genome size and cell size. The proteome composition of the enlarged?genome strains will be studied to unravel how the expression of a class of proteins for cellular reproduction differentially responds to various growth inhibitions. The proposed research will provide increased clarity of the mechanisms underlying cell size, the cell cycle and proliferation, one of the most basic processes critically and clinically important to human life.